PROJECT SUMMARY Recent development of transcriptome-wide approaches to detect modified nucleotides has revealed an expanding repertoire of modified nucleotides in messenger RNA. RNA modifications represent a new layer of eukaryotic gene regulation. Pseudouridine (?) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated in different conditions. However, the endogenous functions of ? in mRNA are not currently understood. We have shown that nascent pre-mRNA is pseudouridylated co-transcriptionally at thousands of locations by multiple Pseudouridine Synthases (PUSs). We found that pre-mRNA ?s are enriched in regions important for splicing regulation and overlap splicing factor binding sites. Genetic manipulation of ? synthases leads to widespread alternative splicing and individual ? directly affect splicing in vitro. PUSs have been linked to a wide range of diseases, including cancer. High expression of pre-mRNA ? synthases correlates with decreased survival in hepatocellular carcinoma cells (HCC) and rewiring of gene expression by alternative splicing is important for HCC pathology. NIH K99/R00 Pathway to Independence Award seeks to define PUS-dependent alternative splicing networks in HCC cells (Aim 1), identify molecular mechanisms by which ?s regulates pre-mRNA splicing (Aim 2) and investigate mechanisms by which ? is deposited in pre-mRNA co-transcriptionally (Aim 3). During the mentored phase of this award Dr. Martinez will identify PUS-dependent alternative splicing in HepG2 cells by sequencing based methods for ? detection and alternative splicing analysis. splicing Dr. Martinez will determine the effect of ? on the binding of candidate ?-sensitive factors, whose binding sites are enriched for overlapping ? locations, using biochemical approaches. During the independent phase, Dr. Martinez will define the HCC specific pre-mRNA ? landscape and identify PUS-dependent HCC specific alternative splicing events by profiling ?, and corresponding splicing changes in primary normal and HCC cells. Her elucidation of mechanisms of ?-mediated splicing regulation in HCC cells will uncover potential disease related modes of splicing regulation. During the K99 and R00 portion of the award Dr. Martinez will determine how ? synthases are recruited to nascent pre-mRNA by investigating mechanisms of recruitment to chromatin and association with Pol II by a combination of chromatin- and co- immunoprecipitation experiments and measuring extent of pseudouridylation in relation to transcription dynamics using a novel sequencing approach. The pathology of disease-linked ? synthases may be due to dysregulation of their pre-mRNA targets. Therefore, defining and characterizing the effect of PUS-dependent pre-mRNA ?s in pre-mRNA processing will help explain how these enzymes contribute to disease. The Pathway to Independence Award will allow Dr. Martinez to gain the necessary expertise (e.g. technology development and computational approaches to discover RNA modifications, techniques to study transcriptional coupling) and professional skills (e.g. mentoring and management) to become a competitive candidate for an academic position at a major research university.